Cellular Structural Fluorescence Staining and Multichannel Imaging
Cellular Structural Fluorescence Staining and Multichannel Imaging
Cellular fluorescence staining converts cell morphology and subcellular structures into observable and quantifiable spatial signals through selective labeling of the nucleus, F-actin, microtubules, plasma membrane, and organelles. The key to experimental design is not simply increasing the number of fluorescent colors, but establishing non-interfering labeling combinations according to the target structure, sample state, fixation and permeabilization conditions, dye spectra, and microscope channel configuration, thereby enabling accurate analysis of cell spreading, cytoskeletal remodeling, organelle distribution, and structural colocalization.
Keywords: fluorescence staining; multichannel imaging; F-actin; phalloidin; nuclear staining; organelle staining; confocal microscopy
1 Technical Basis of Cellular Structural Fluorescence Staining
1.1 Major Types of Structural Labeling
Cellular structural fluorescence labeling mainly includes nucleic acid dyes, cytoskeletal probes, membrane dyes, organelle probes, and antibody-mediated immunofluorescence. Different systems recognize different targets: nuclear dyes mainly bind DNA, phalloidin selectively binds polymerized Actin, microtubules are usually visualized using Tubulin antibodies or corresponding probes, and mitochondria, lysosomes, endoplasmic reticulum, and Golgi can be localized according to specific membrane structures, organelle microenvironments, or marker proteins.
1.2 Fixed-Cell and Live-Cell Staining
(1) Fixed-Cell Staining
Fixation stabilizes the overall cellular structure and allows phalloidin, antibodies, and other probes to enter the cell after permeabilization. Fixed samples are suitable for localization of F-actin, Tubulin, and many proteins and also facilitate prolonged confocal acquisition and multichannel imaging. However, fixation itself may alter membrane structures, the state of some organelles, and protein conformations.
(2) Live-Cell Staining
Live-cell staining requires probes to enter cells or localize to target structures while maintaining cellular integrity and is commonly used for dynamic observation of the cell membrane, mitochondria, lysosomes, and some nucleic acids. Its advantage is the ability to continuously track structural changes, but probe concentration, incubation time, phototoxicity, and effects of dyes on cellular physiological status all need to be controlled.
1.3 Single-Channel and Multichannel Imaging
Single-channel staining is suitable for evaluating the area, intensity, or morphology of one structure, whereas multichannel imaging can establish spatial relationships between nucleus and cytoplasm, cytoskeleton and membrane, cytoskeleton and organelles, or proteins and organelles. Increasing the number of channels also increases spectral crosstalk, exposure differences, and image-analysis complexity. Therefore, multicolor design should prioritize sufficient spectral separation between channels rather than simply increasing the number of colors.
2 Fluorescence Staining of the Nucleus and Nucleic Acids
2.1 DAPI-Based Nuclear Staining
(1) Staining Characteristics
DAPI binds DNA and produces strong blue fluorescence and is mainly used to visualize nuclei and chromatin structures. Its signal is concentrated in the nucleus and can serve as a spatial reference in multichannel imaging as well as for cell counting, nuclear area, and nuclear morphology analysis.
(2) Major Advantages
DAPI provides clear nuclear contours, and its blue fluorescence is well separated spectrally from 488 nm, orange-red, red, and far-red channels. It is therefore suitable for combination with F-actin, organelle probes, and fluorescent antibodies. Staining workflows for fixed cells and tissues are well established, making DAPI particularly suitable as a basic nuclear counterstain in multicolor immunofluorescence.
(3) Major Limitations
DAPI has limited applicability to intact live cells and is more commonly used for fixed or permeabilized samples. Excessive dye concentration or overexposure can saturate nuclear signals and affect quantification of chromatin condensation, nuclear area, and fluorescence intensity. Therefore, staining and acquisition conditions should remain consistent across treatment groups.
2.2 Hoechst-Based Nuclear Staining
(1) Staining Characteristics
Hoechst dyes mainly bind DNA and produce blue fluorescence, and some members have relatively good cell-membrane permeability, allowing nuclear staining of both fixed and live cells. Staining can reveal nuclear position, nuclear contours, and some changes in chromatin morphology.
(2) Major Advantages
Compared with DAPI, some Hoechst dyes are more suitable for live-cell applications and can be used for live-cell counting, nuclear localization, and dynamic imaging. Their blue channel is well separated from commonly used fluorescent regions such as FITC/LumiDye™ 488, 594, and 647, facilitating three- or four-color imaging systems.
(3) Major Limitations
Hoechst signals are also affected by dye concentration, incubation time, and exposure parameters, and high concentrations or prolonged treatment may increase cytotoxicity. In live-cell dynamic studies, changes in staining intensity should not be directly interpreted as changes in DNA content.
2.3 PI Nucleic Acid Staining
(1) Staining Characteristics
Propidium Iodide (PI) binds nucleic acids and produces red fluorescence, but intact live-cell membranes usually restrict PI entry. It is therefore commonly used for dead-cell identification, evaluation of cell-membrane integrity, and DNA-content detection after fixation or permeabilization.
(2) Major Advantages
PI produces strong signals and has well-established applications. It can be combined with some blue or green fluorescence channels for cell viability and nucleic acid analysis. Because its entry strongly depends on membrane integrity, PI has clear interpretive value in dead-cell identification and membrane-damage assessment.
(3) Major Limitations
PI is not suitable as a routine nuclear counterstain for intact live cells. Its emission lies in the red spectral region and may overlap with Rhodamine, 594, and some red organelle probes, so channels need to be planned in advance in multicolor experiments.
2.4 Interpretation of Nuclear Staining
Nuclear fluorescence can be used for cell counting, nuclear area, nuclear circularity, chromatin condensation, multinucleated cells, and nuclear fragmentation analysis. When quantitatively comparing different treatment groups, dye concentration, microscope exposure, and image thresholds should remain consistent to avoid interpreting intensity changes caused by technical parameters as changes in DNA content or cellular state.
3 F-actin and Cytoskeletal Fluorescence Staining
3.1 Structural Basis of the Actin Cytoskeleton
Actin mainly exists as globular G-actin and polymerized F-actin. F-actin can further form stress fibers, cortical Actin, lamellipodia, filopodia, and other structures involved in cell spreading, migration, contraction, adhesion, mechanosensing, and maintenance of cell morphology. Therefore, the spatial organization of F-actin provides cytoskeletal information distinct from total Actin expression levels.
3.2 Phalloidin Labeling
(1) Binding Characteristics
Phalloidin has high affinity for polymerized F-actin and, after fluorescent labeling, can directly visualize cell edges, stress fibers, cellular protrusions, and other Actin-enriched regions. Because it mainly recognizes F-actin, it cannot serve as a direct quantitative indicator of total Actin or G-actin.
(2) Major Advantages
Fluorescent phalloidin does not require primary or secondary antibodies, reducing species restrictions and secondary-antibody cross-reactivity in immunofluorescence. Green, orange-red, red, or far-red fluorescent labels can be selected according to existing channels. Its structural visualization is generally direct for studies of cell spreading, migration, and cytoskeletal remodeling.
(3) Major Limitations
Phalloidin has limited ability to enter intact cell membranes, so fixed-cell experiments generally require moderate permeabilization. Insufficient fixation may destabilize cytoskeletal structures, whereas excessive permeabilization may cause membrane disruption, loss of soluble proteins, or deformation of cell edges. Therefore, staining quality is affected by both the probe and pretreatment conditions.
3.3 Tubulin Immunofluorescence Labeling
(1) Labeling Characteristics
Microtubules are formed by polymerization of α/β-Tubulin and can be visualized using antibodies against α-Tubulin, β-Tubulin, or specific post-translationally modified forms of Tubulin. Staining can reveal the cytoplasmic microtubule network, microtubule-organizing centers, mitotic spindles, and some ciliary structures.
(2) Major Advantages
Tubulin antibodies can be selected according to the research objective to detect total Tubulin or specific modification states such as acetylated Tubulin. This enables evaluation of both overall microtubule structure and stabilized or functionally distinct microtubule states. Combined with F-actin staining, the two major cytoskeletal systems can be observed simultaneously.
(3) Major Limitations
Microtubules are relatively sensitive to fixation conditions, and different fixation methods may markedly affect structural preservation and antigen accessibility. Immunofluorescence also depends on antibody specificity, secondary-antibody selection, and permeabilization conditions. Therefore, microtubule fragmentation, disappearance, or increased background does not necessarily represent a true biological change.
3.4 Combined Imaging of F-actin and Microtubules
F-actin mainly participates in cortical structures, stress fibers, and formation of the migration front, whereas microtubules generally extend from the microtubule-organizing center toward the cell periphery. Combined staining can evaluate establishment of cell polarity, migration direction, drug-induced cytoskeletal remodeling, and coordinated changes between the two cytoskeletal systems during mitosis.
Table 1 Comparison of Major Cytoskeletal Labeling Methods
Target Structure | Common Method | Major Advantages | Major Limitations | Applicable Research |
F-actin | Fluorescent phalloidin | Direct, clear structure, antibody-free | Does not reflect G-actin | Stress fibers, spreading, migration |
Total Actin | Actin antibody | Evaluates protein expression and localization | Cannot independently distinguish polymerization state | Actin expression and localization |
α/β-Tubulin | Immunofluorescence | Clear microtubule structure | Dependent on antibodies and fixation conditions | Microtubule networks, cell polarity |
Modified Tubulin | Specific antibodies | Distinguishes different microtubule states | Strong antibody dependence | Microtubule stability, cilia research |
4 Cell Membrane, Cell Outline, and Cell Tracking
4.1 Lipophilic Membrane Dyes
(1) Labeling Characteristics
Lipophilic dyes such as DiI, DiO, DiD, and DiR insert into lipid bilayers to generate membrane fluorescence signals without recognizing specific membrane proteins. Different dyes cover green, orange-red, red, and near-infrared spectral regions and can be used for cell boundaries, membrane morphology, and cell-population labeling.
(2) Major Advantages
Lipophilic membrane dyes are relatively straightforward to use and can produce continuous cell-membrane outlines, making them suitable for observing cell edges, cell-cell contacts, membrane protrusions, and some migration processes. Different spectral versions can also distinguish multiple cell populations or be combined with nuclear, cytoskeletal, and organelle signals.
(3) Major Limitations
Membrane dyes may redistribute through membrane movement, membrane exchange, or endocytosis and therefore cannot be interpreted directly as localization of a specific membrane protein. Long-term tracking also requires consideration of dye transfer, signal dilution, and changes in cellular state.
4.2 Membrane Staining and F-actin Staining
Plasma-membrane outlines and cortical F-actin are spatially close but represent different structures. Combined staining can distinguish changes in membrane morphology from Actin repolymerization and is suitable for studies of cell spreading, membrane protrusion formation, migration fronts, and cell-cell contacts.
4.3 Cell-Tracking Dyes
(1) Labeling Characteristics
Cell-tracking dyes are used to establish persistent fluorescent labeling of specific cell populations and can be applied to migration, coculture, cell-population discrimination, and in vitro or in vivo tracking. Their core readouts are the location, migration, and persistence of labeled cells rather than a specific subcellular structure.
(2) Major Advantages
Stable cell-tracking signals can be combined with nuclear staining, F-actin, immunofluorescence, or organelle staining to distinguish cells of different origins in complex systems. In coculture, cell migration, and cell-transfer experiments, a cell-identity channel can substantially improve spatial interpretation.
(3) Major Limitations
Dyes are diluted during cell division, and some dyes may also be affected by cellular metabolism, membrane exchange, or efflux. Long-term tracking therefore requires consideration of signal-retention time, cytotoxicity, and labeling stability.
5 Organelle Fluorescence Labeling Systems
5.1 Mitochondrial Structural Labeling
(1) Labeling Characteristics
Mitochondrial structural staining is mainly used to visualize mitochondrial networks, perinuclear and peripheral distribution, length, branching, and fragmentation. Mitochondria-localizing fluorescent probes can be used, or mitochondrial structures can be visualized by immunofluorescence of marker proteins such as TOMM20.
(2) Major Advantages
Structural labeling can directly reflect mitochondrial fusion, fission, and spatial rearrangement and is more suitable for network-morphology analysis than total whole-cell fluorescence intensity. When combined with the cytoskeleton, nucleus, or other organelles, it can also evaluate mitochondrial transport and spatial localization.
(3) Major Limitations
Accumulation of some so-called “mitochondrial probes” is actually dependent on membrane potential. Therefore, not all mitochondrial fluorescence signals can be used directly for structural quantification. Before analysis, it is necessary to determine whether the probe is structure-localizing or function-dependent.
5.2 Mitochondrial Membrane-Potential Probes
(1) Detection Characteristics
Probes such as JC-1 and JC-10 mainly exhibit different fluorescence behaviors according to changes in mitochondrial membrane potential and are used to evaluate mitochondrial functional status and cellular injury rather than simply visualize mitochondrial structure.
(2) Major Advantages
Changes in membrane potential occur during many forms of cellular stress, drug treatment, and apoptosis. These probes can therefore be used for early detection of mitochondrial dysfunction and can be analyzed by flow cytometry or fluorescence microscopy.
(3) Major Limitations
A decrease in membrane potential directly changes probe signals, so reduced fluorescence or changes in color ratio cannot be interpreted as a reduction in mitochondrial number and cannot independently determine fusion, fission, or network fragmentation. These results need to be distinguished from structural labeling or other mitochondrial functional indicators.
5.3 Lysosomal Acidic-Environment and Structural Labeling
(1) Labeling Characteristics
Probes such as Lyso-Tracker mainly accumulate according to the acidic environment of organelles and can visualize the number, area, and intracellular distribution of lysosomes and related acidic vesicles.
(2) Major Advantages
These probes are suitable for live-cell observation and can evaluate perinuclear aggregation, cytoplasmic dispersion, and spatial relationships between lysosomes and other organelles. They provide relatively direct spatial information for studies of cellular stress, autophagy, and lysosomal transport.
(3) Major Limitations
Signal intensity is affected by luminal pH. Therefore, reduced fluorescence may result from either decreased lysosome number or impaired acidification. When structural changes need to be determined, lysosomal markers such as LAMP1/LAMP2 can be used for validation.
5.4 Endoplasmic Reticulum Fluorescence Labeling
(1) Labeling Characteristics
ER-Tracker and other endoplasmic-reticulum-targeting probes can visualize the ER membrane network surrounding the nucleus and extending into the cytoplasm and are used to analyze ER morphology and subcellular distribution.
(2) Major Advantages
Chemical probes can rapidly visualize the overall endoplasmic-reticulum network and are suitable for live-cell imaging, ER rearrangement, and organelle-contact studies. When combined with antibodies against specific ER proteins, overall structure and target-molecule localization can be further distinguished.
(3) Major Limitations
The endoplasmic reticulum is a highly dynamic membranous structure, and fixation, permeabilization, and dye treatment may all alter its morphology. Different probes also differ in localization mechanisms and live-cell compatibility, so fluorescence intensity alone cannot be used to determine ER abundance or stress levels.
5.5 Golgi Fluorescence Labeling
(1) Labeling Characteristics
The Golgi apparatus is usually located near the nucleus and shows a certain degree of polarity. Golgi-targeting probes or fluorescent ceramide-based labels can visualize its perinuclear distribution, structural integrity, and repositioning.
(2) Major Advantages
Golgi localization is closely associated with cell polarity and vesicular transport. When combined with microtubules, F-actin, or nuclear staining, it can be used to analyze Golgi orientation during migration and rearrangement of the endomembrane system.
(3) Major Limitations
Golgi dispersion or changes in perinuclear signals may reflect true structural rearrangement but can also be affected by fixation, cell cycle, or probe trafficking. Some ceramide-based probes may also enter other membrane systems, so results need to be interpreted together with Golgi-specific marker proteins.
Table 2 Key Interpretation Points for Major Organelle Fluorescence Labeling
Labeled Structure | Major Observation Indicators | Major Advantages | Interpretation Limitations |
Mitochondria | Network length, branching, distribution, membrane potential | Structural and functional evaluation can be performed separately | Membrane-potential probes cannot directly represent quantity |
Lysosomes | Number, area, intracellular distribution | Clear visualization of acidic organelles | Signal is affected by pH |
Endoplasmic reticulum | Network morphology, perinuclear distribution | ER rearrangement can be observed | Fixation and permeabilization may affect morphology |
Golgi | Perinuclear localization, polarity | Suitable for migration and transport studies | Structural rearrangement needs to be distinguished from changes in probe distribution |
6 Single-, Dual-, and Multichannel Staining Design
6.1 Single-Structure Staining
Single labeling is suitable for establishing staining conditions, evaluating probe specificity, and performing quantitative analysis of a single structure. New experiments can first use single staining to determine working concentration, exposure parameters, and background levels before proceeding to multicolor combinations, thereby reducing subsequent channel crosstalk and complexity in determining signal origin.
6.2 F-actin and Nuclear Dual Staining
F-actin and nuclear dual staining is a basic combination with relatively high information density in cellular structural imaging: nuclear signals are used for cell localization, counting, and nuclear morphology evaluation, whereas F-actin reveals cell outlines, spreading area, stress fibers, and cellular protrusions. It is suitable for cell adhesion, migration, drug treatment, and morphological screening. One-step phalloidin/nuclear blue dual-staining kits can reduce the need to prepare probes separately and are suitable for standardized routine imaging. Independent fluorescent phalloidin provides greater flexibility in spectral combinations and is more suitable for complex multicolor imaging with DAPI/Hoechst, fluorescent antibodies, or organelle probes.
6.3 Three-Color and Higher-Order Combinations
A typical three-color design can use blue nuclear staining, a green target protein, and red or far-red F-actin. Alternatively, F-actin can be assigned to the green channel and antibody signals to red and far-red regions. For low-abundance or weak-signal targets, channels with lower background and better instrument detection efficiency should be prioritized rather than assigning a specific cell structure to a fixed color.
6.4 Multicolor Channel Selection
Table 3 Characteristics of Common Fluorescence Regions in Multichannel Imaging
Spectral Region | Representative Labels | Major Advantages | Major Limitations | Applicable Combinations |
Blue | DAPI, Hoechst, nuclear blue | Separated from most visible-light channels | Mainly occupies the nuclear-staining channel | Nucleus + 488 + 594/647 |
Green | FITC, 488-type | High instrument compatibility | Some samples show relatively high autofluorescence | Routine dual-label and three-color imaging |
Orange-red | Rhodamine, 555-type | Strong visual contrast | Spectrally close to some orange-red dyes | Nucleus + F-actin |
Red | 594-type | Good separation from 488 | Occupies a commonly used red antibody channel | Three-color IF |
Far-red | 633/647-type | Autofluorescence is usually relatively low | Requires corresponding laser and filters | Multiplex IF, confocal microscopy |
Long-wavelength far-red | 680-type | Frees commonly used visible-light channels | Compatibility with conventional microscopes needs confirmation | High-channel-count imaging |
7 Effects of Fixation and Permeabilization on Cellular Structure Preservation
7.1 Paraformaldehyde Fixation
Paraformaldehyde maintains overall cellular morphology through protein crosslinking and is a common fixation method for F-actin and most immunofluorescence experiments. It provides a relatively balanced preservation of cell outlines and cytoskeletal morphology, but overfixation may reduce accessibility of some antigens. Therefore, multicolor experiments need to consider both structural preservation and subsequent antibody recognition.
7.2 Alcohol Fixation
Alcohols such as Methanol achieve fixation through protein precipitation and dehydration and are suitable for some microtubule antigens but may alter membrane systems and some Actin structures. If the experiment primarily evaluates F-actin morphology, membrane outlines, or membranous organelles, alcohol fixation should not automatically be regarded as equivalent to PFA fixation.
7.3 Triton-Based Permeabilization
Nonionic surfactants such as Triton X-100 can increase cell-membrane permeability and allow phalloidin and antibodies to enter the cytoplasm and nucleus. Excessive permeabilization may cause membrane disruption, loss of soluble proteins, or deformation of cell edges. Therefore, cytoplasmic, nuclear, and membrane-associated targets generally require different permeabilization strengths.
7.4 Mild Permeabilization Systems
Relatively mild permeabilization conditions can be used when membrane-structure preservation and intracellular probe entry need to be balanced. Their advantage is reduced structural loss caused by strong membrane extraction, but penetration of deep structures or high-molecular-weight probes may be lower. Therefore, treatment conditions need to be determined according to target location and probe type.
Table 4 Major Differences Among Fixation and Permeabilization Conditions
Condition | Major Advantages | Major Limitations | Preferred Applications |
PFA fixation | Good preservation of overall morphology and F-actin | Overfixation may affect antigens | F-actin, IF, multicolor staining |
Methanol fixation | Rapid and suitable for some Tubulin detection | May alter membrane and Actin structures | Specific microtubule antigens |
Triton-based permeabilization | Strong permeabilization capacity | Excessive treatment may cause structural loss | Cytoplasmic and nuclear antigens |
Mild permeabilization systems | Relatively small effect on membrane structures | Penetration of deeper structures may be insufficient | Membrane-associated and some intracellular staining |
8 Signal Interference in Multichannel Imaging
8.1 Spectral Crosstalk
When the emission ranges of two fluorophores overlap substantially, a strong signal from one channel may enter another detection window and produce false colocalization. Multicolor experiments should use single-stained samples to confirm the actual channel distribution of each fluorescent signal before determining lasers, filters, and detection ranges.
8.2 Autofluorescence
Intrinsic cellular and tissue components can generate background fluorescence, and fixation can also increase some forms of autofluorescence. The green spectral region is relatively susceptible to autofluorescence from some biological samples. Therefore, in high-background samples, critical weak signals can be preferentially assigned to red or far-red regions.
8.3 Imbalance in Fluorescence Intensity
Strong nuclear staining or high-abundance F-actin signals can occupy a large portion of the image dynamic range, whereas low-abundance proteins may require higher gain or longer exposure. Acquisition parameters should be independently set for each channel, avoiding adjustment of the original quantitative relationship merely to achieve visually balanced colors.
8.4 Photobleaching and Phototoxicity
Repeated scanning of fixed samples can cause fluorescence photobleaching, whereas live cells additionally face phototoxicity. Multichannel confocal experiments should control laser power, exposure time, and number of scans. Fixed samples can be combined with antifade mounting systems to improve signal stability during imaging and storage.
9 Quantification and Interpretation of Cellular Structural Fluorescence Images
Quantification of fluorescence structural imaging should distinguish between “signal intensity” and “spatial structure.” Total fluorescence intensity mainly reflects the overall level of the probe or labeling signal, whereas area, length, branching, orientation, perinuclear distribution, and colocalization reflect structural organization. For objects with clear spatial morphology such as F-actin, mitochondria, and Golgi, comparison of average fluorescence intensity alone is generally insufficient to support conclusions regarding structural remodeling.
9.1 F-actin Remodeling
F-actin experiments can analyze stress-fiber number, fiber orientation, cortical Actin proportion, cell spreading area, and protrusion formation. Increased total fluorescence intensity does not necessarily indicate enhanced stress fibers. Redistribution of Actin from the inner cytoplasm to the cell periphery can also produce pronounced morphological changes while total signal remains largely unchanged.
9.2 Cell Area and Morphology
Phalloidin or membrane labeling can clearly define the cell outline of most adherent cells and can be used to calculate area, aspect ratio, circularity, and polarity. In migration, EMT, and cell-adhesion experiments, morphological parameters should be interpreted together with migration capacity, adhesion status, or related molecular markers.
9.3 Spatial Distribution of Organelles
Perinuclear/peripheral distribution of mitochondria, lysosomes, and Golgi can reflect changes in organelle transport and cell polarity. Analysis can include number, area, network length, branching, distance from the nucleus, and spatial distribution of organelles rather than comparing only whole-cell average fluorescence intensity.
9.4 Colocalization
Colocalization analysis is used to evaluate spatial overlap between two fluorescent signals, but pixel overlap does not indicate direct molecular interaction. Interpretation should consider optical resolution, channel crosstalk, background thresholds, and random overlap.
10 Common Abnormalities and Experimental Troubleshooting
Table 5 Common Abnormalities in Cellular Fluorescence Staining
Experimental Observation | Major Related Factors | Priority Troubleshooting Direction |
Weak F-actin signal | Phalloidin concentration, permeabilization, fixation | Dye concentration, incubation time, permeabilization conditions |
Fragmented stress fibers | Excessive fixation/permeabilization | Fixative, permeabilizing-agent concentration, and treatment time |
Blurred cell edges | Insufficient cytoskeletal preservation | Fixation method, cellular state |
Saturated nuclear signal | Excessive nuclear dye, excessive exposure | Dye concentration and exposure |
High green background | Autofluorescence, excessive dye | Washing conditions, exposure, or channel selection |
Multichannel bleed-through | Spectral overlap | Single-stain controls, detection windows |
Weak far-red signal | Mismatched laser/filter | Instrument channels and detector settings |
Abnormal organelle signal | Probe affected by membrane potential or pH | Distinguish structural changes from functional changes |
Excessive colocalization | Crosstalk, overly low threshold | Single-stain controls, threshold, and spectral settings |
11 Products Related to Cellular Structural Fluorescence Staining and Multichannel Imaging
11.1 F-actin and Microtubule Staining Products
Catalog # | Product Name | Grade & Purity | Main Application |
One-Step LumiDye™ 488 Phalloidin/Nuclear Blue Dual Staining Kit | BioReagent, biological stain, for microscopy, sterile, ready-to-use | Dual staining of green F-actin and blue nuclei | |
One-Step LumiDye™ 555 Phalloidin/Nuclear Blue Dual Staining Kit | ready-to-use, sterile, for microscopy, biological stain, BioReagent | Dual staining of orange-red F-actin and blue nuclei | |
One-Step LumiDye™ 594 Phalloidin/Nuclear Blue Dual Staining Kit | BioReagent, biological stain, for microscopy, sterile, ready-to-use | Dual staining of red F-actin and blue nuclei | |
One-Step LumiDye™ 633 Phalloidin/Nuclear Blue Dual Staining Kit | BioReagent, biological stain, for microscopy, sterile, ready-to-use | Dual staining of far-red F-actin and blue nuclei | |
One-Step LumiDye™ 680 Phalloidin/Nuclear Blue Dual Staining Kit | BioReagent, biological stain, for microscopy, sterile, ready-to-use | Dual staining of long-wavelength far-red F-actin and blue nuclei | |
One-Step Rhodamine Phalloidin/Nuclear Blue Dual Staining Kit | BioReagent, biological stain, for microscopy, sterile, ready-to-use | Dual staining of Rhodamine-F-actin and nuclei | |
Rhodamine-Labeled Phalloidin (Orange-Red) | BioReagent, biological stain, for microscopy, sterile, Ex: 540-546nm; Em: 565-575nm | Orange-red fluorescent staining of F-actin | |
FITC-Labeled Phalloidin | BioReagent, biological stain, for microscopy, sterile, Ex:495-496nm; Em:513-516nm | Green fluorescent staining of F-actin | |
LumiDye™ 555-Labeled Phalloidin (Orange-Red) | BioReagent, biological stain, for microscopy, sterile, Ex:555nm; Em:565nm | Orange-red channel labeling of F-actin | |
LumiDye™ 594-Labeled Phalloidin (Red) | BioReagent, biological stain, for microscopy, sterile, Ex:581nm;Em:609nm | Red-channel labeling of F-actin | |
LumiDye™ 647-Labeled Phalloidin (Far-Red) | BioReagent, biological stain, for microscopy, sterile | Far-red channel labeling of F-actin | |
Phalloidin–Atto 594 | Suitable for fluorescence analysis, ≥90% (HPLC) | Red fluorescent labeling of F-actin | |
Phalloidin-TRITC | ≥97% | Orange-red fluorescent staining of F-actin | |
Phalloidin from Amanita phalloides | Moligand™, ≥90% | F-actin binding and cytoskeletal mechanism research | |
alpha Tubulin Mouse mAb (AF405) | ExactAb™, Validated, 0.5 mg/mL | Blue-violet channel fluorescent labeling of microtubules | |
alpha Tubulin Mouse mAb (AF750) | ExactAb™, Validated, 0.5 mg/mL | Far-red/near-infrared channel labeling of microtubules | |
beta Tubulin Mouse mAb (AF647) | ExactAb™, Validated, Azide Free, 0.5 mg/mL | Far-red fluorescent labeling of β-Tubulin |
11.2 Nuclear and Nucleic Acid Staining Products
Catalog # | Product Name | Grade & Purity | Main Application |
DAPI Staining Solution | BioReagent, for microscopy, sterile-filtered, Suitable for Immunofluorescence (IF), 1.0 mg/mL | Blue fluorescent staining of DNA and cell nuclei | |
DAPI Staining Solution (Ready-to-Use) |
| Ready-to-use nuclear staining and multicolor imaging | |
Hoechst 33342 Fluorescent Dye | ≥98% | Nuclear staining of live and fixed cells | |
Bisbenzimide H 33258 Fluorochrome, Trihydrochloride | Membrane-permeable, adenine-thymine-specific fluorescent stain. | DNA, chromosome, and cell-nucleus staining | |
Propidium Iodide (PI) Staining Solution (Ready-to-Use) | BioReagent, for microscopy, ready-to-use, sterile-filtered, Suitable for Immunofluorescence (IF) | Detection of nucleic acids, dead cells, and DNA content |
11.3 Cell Membrane, Cell Outline, and Cell Tracking Products
Catalog # | Product Name | Grade & Purity | Main Application |
DiO Plus (Cell Membrane Green Fluorescent Probe Upgrade) |
| Green plasma membrane staining and long-term cell tracking | |
CytoMBrite™ Cell Membrane Orange-Red Fluorescent Probe (DiI) |
| Orange-red cell membrane staining and tracking of migration and adhesion | |
DiD (Cell Membrane Red Fluorescent Probe) |
| Red/far-red cell membrane imaging | |
DiR' [DiIC18 (7)], for Membrane Staining | ≥95% | Near-infrared membrane staining and cell tracking | |
DiIC16 (3) (Cell Membrane Fluorescent Probe) | BioReagent, for microscopy, biological stain, suitable for fluorescence analysis, ≥95% | Labeling of live-cell membranes and hydrophobic structures | |
CellTracker Red CMTPX | BioReagent, ≥80% (HPLC) | Cell localization, migration, and long-term tracking | |
aladdin™ Red Fluorescent Cell Linker Kit |
| Stable cell membrane labeling and cell tracking |
11.4 Mitochondrial, Lysosomal, Endoplasmic Reticulum, and Golgi Fluorescent Products
Catalog # | Product Name | Grade & Purity | Main Application |
Mitochondrial Membrane Potential Assay Kit (JC-1) |
| Evaluation of mitochondrial membrane potential and early cellular injury | |
Mitochondrial Membrane Potential Assay Kit (JC-10) | BioReagent | Fluorescent detection of mitochondrial membrane potential | |
Lyso-Tracker Red (Lysosome Red Fluorescent Probe) |
| Red labeling of lysosomes in live cells | |
Lysosome Red Probe (Aggregation-Induced Emission) | BioReagent, biological stain, for microscopy, ≥98% (HPLC), 10 mM in DMSO | Red/far-red lysosome imaging in live cells | |
ER-Tracker Green |
| Green fluorescent staining of the endoplasmic reticulum in live cells | |
ER-Tracker Blue-White DPX |
| Blue-white fluorescent labeling of the endoplasmic reticulum | |
AIE-ER | ≥98% | AIE fluorescence imaging of the endoplasmic reticulum | |
NBD C6-Ceramide |
| Golgi staining in live and fixed cells | |
Gol-NTR | ≥99% | Research on Golgi-targeted fluorescent probes |
11.5 Antifade Mounting Products
Catalog # | Product Name | Grade & Purity | Main Application |
Antifade Mounting Medium |
| Mounting of fluorescent cell and tissue samples and retardation of photobleaching | |
Enhanced Antifade Mounting Medium | BioReagent, Suitable for Fluorescence Analysis, Suitable for Immunofluorescence (IF) | Antifade mounting for multicolor IF and fluorescent samples |
The reliability of cellular structural fluorescence imaging depends on combined control of probe specificity, structural preservation, and spectral configuration. Different dyes and probes should be selected according to the target structure, sample state, and instrument channels, with clear distinction as to whether their signals represent structural localization, functional status, or cell identity. Combined with single-stain controls, consistent acquisition parameters, and structure-based image quantification, this approach can effectively distinguish genuine spatial changes from signal differences caused by staining, spectral crosstalk, or imaging conditions.
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